Experimental Study Of The Effects Of Composite Pseudostellaria Granule On Denervated Muscular Atrophy

Purpose: To investigate the retarding mechanism of Composite Pseudostellaria Granule (CPG) on denervated skeletal muscular atrophy, and provide data for the practical use of CPG, by studying the effects of CPG on denervated muscular atrophy.MethodsrThe model of denervated skeletal musle was established by cutting off the right tibial nerve of Sprague Dawley rats.54 model Sprague Dawley rats were randomly divided into experimental group and control group, and administered respectively with CPG and normal saline every day after surgery. Samples were taken from the gastrocnemius and all the indexes were tested in 2, 4, 6 weeks of operative intervals respectively. The muscle wet weight was weighed by electron balance.The total protein content of atrophic muscle was measured by biuret reaction. The diameter and cross section area of muscle cell were measured with HE staining. The collagen fiber area, and collagen/muscle cell ratio of area were studied with Sirius Red staining. The apoptotic cells in denervated skeletal muscle were examined with TUNEL. The expressions of actin and myosin, apoptosis associated genes Fas and Bcl-2 were studied with immunohistochemical technique. The change of ultrastructure was oberserved with transmission electron microscope.Results: (1) After denervation, the gastrocnemius muscle of every rat was obviously atrophic. The musle wet weight rapidly reduced in the control group, while it reduced more slowly in the experimental group. The experimental group was significantly greater than the control group in the muscle wet weight.(2) The total protein content of atrophic skeletal muscle reduced progressively as denervation prolonged. The total protein content in the experimental group was significantly greater than that in the control group. (3) The diameter and cross section area of the muscle fibers decreased gradually in both groups after denervation, while the experimental group was remarkably larger than the control group in the two parameters.(4) The collagen fiber content increased in the denervated muscle afteroperation. The proliferation of collagen in the control group was more obvious than that in the experimental group. The collagen fiber area , and collagen/muscle cell ratio of area were higher significantly in the control group than that in the experimental group. (5) The expressions of actin and myosin reduced in the denervated muscle along with the denervated time, while their expressions in the experimental group were greater than those in the control group. (6) Apoptotic nucleus was not seen in normal skeletal muscle, while it was obviously seen in the denervated skeletal muscle.With elongation of denervation time, the number of apoptotic nucleus increased. The number of apoptotic nucleus in the experimental group was remarkably less than that in the control group. The apoptosis associated gene Fas was expressed highly and Bcl-2 was expressed lowly in atrophic musle after denervation. The expression of Fas protein was significantly downregulated and the expression of Bcl-2 protein was significantly upregulated in the experimental group, compared to the control group. (7) No classic apoptotic body was seen in the denervated skeletal muscle, but morphologic changes of early stage in apoptotic cell could be seen under electron microscope, such as the aggregation of chromosome, the expansion of nucleic cistern and the contraction of nucleus. Compared to the control group, less degenerated nucleus, minor mitochondria edema and sarcoplasmic reticulum enlargement, more capillary , less interstitial fibroblast and collagen fibers, better oriented myomere and myofilament were seen in the experimental group with transmission electron microscope. Conclusions:1. Apoptosis is closely related to denervated muscular atrophy. The increase of Fas gene expression and decrease of Bcl-2 gene expression play an important role in apoptosis of skeletal muscle cell.2. CPG can effectively retard denervated skeletal muscular atrophy. The retarding mechanism may be as follows:(1) CPG ca...